Three Babesia Species in Ixodes Ricinus Ticks from Migratory Birds in Sweden Peter Wilhelmsson1,2*†, Olga Pawełczyk1,3†, Thomas G

Wilhelmsson et al. Parasites Vectors (2021) 14:183 https://doi.org/10.1186/s13071-021-04684-8 Parasites & Vectors

RESEARCH Open Access Three Babesia species in Ixodes ricinus ticks from migratory birds in Sweden Peter Wilhelmsson1,2*†, Olga Pawełczyk1,3†, Thomas G. T. Jaenson4, Jonas Waldenström5, Björn Olsen6, Pia Forsberg7 and Per‑Eric Lindgren1,2

Abstract Background: Migratory birds can cross geographical and environmental barriers and are thereby able to facilitate transmission of tick-borne pathogens both as carriers of infected ticks and as reservoirs of pathogenic microorgan‑ isms. Ixodes ricinus is one of the most abundant tick species in the Northern Hemisphere and a main vector of several Babesia species, some which pose a potential threat to human and animal health. At present only two cases of overt babesiosis in humans have so far been reported in Sweden. To better understand the potential role of birds as dis‑ seminators of zoonotic Babesia protozoan parasites, we investigated the presence of Babesia species in ticks removed from migratory birds. Methods: Ticks were collected from birds captured at Ottenby Bird Observatory, south-eastern Sweden, from March to November 2009. Ticks were molecularly identifed to species, and morphologically to developmental stage, and the presence of Babesia protozoan parasites was determined by real-time PCR. Results: In total, 4601 migratory birds of 65 species were examined for tick infestation. Ticks removed from these birds have previously been investigated for the presence of Borrelia bacteria and the tick-borne encephalitis virus. In the present study, a total of 1102 ticks were available for molecular analysis of Babesia protozoan parasites. We found that 2.4% of the ticks examined, all I. ricinus, were positive for mammal-associated Babesia species. Out of all Babesia-positive samples, Babesia venatorum was the most prevalent (58%) species, followed by Babesia microti (38%) and Babesia capreoli (4.0%). B. venatorum and B. capreoli were detected in I. ricinus larvae, whereas B. microti was only present in I. ricinus nymphs. This supports the view that the two frst-mentioned species are vertically (transovarially) transmitted in the tick population, in contrast to B. microti. The largest number of Babesia-infected ticks was removed from the common redstart (Phoenicurus phoenicurus) and European robin (Erithacus rubecula). Conclusions: This study reveals that Babesia protozoan parasites are present in ticks infesting migratory birds in south-eastern Sweden, which could potentially lead to the dissemination of these tick-borne microorganisms into new areas, thus posing a threat to humans and other mammals. Keywords: Babesia capreoli, Babesia microti, Babesia venatorum, Ixodes ricinus, Migratory birds, Tick-borne diseases, Sweden

Background Te role of migratory birds as hosts of disease vectors such as ticks and of potentially human pathogenic micro- *Correspondence: [email protected] †Peter Wilhelmsson and Olga Pawełczyk contributed equally to this work organisms has been increasingly recognized. Birds can 1 Division of Infammation and Infection, Department of Biomedical cross geographical and environmental barriers and take and Clinical Sciences, Linköping University, Linköping, Sweden part in the dispersal of bacteria, viruses, and protozoa [1]. Full list of author information is available at the end of the article Seasonal migration is especially pronounced in northern

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Europe, where a large part of the avifauna migrate, either serious mode of accidental transmission of B. microti to milder regions in western Europe or the Mediter- [26]. Human B. microti infection by blood transfusion ranean region, or long distances to sub-Saharan Africa has also been documented in Europe [24]. or even Asia. Tis means that birds returning in spring In Sweden, Ixodes ricinus is the most abundant tick spe- could carry ticks from regions that better sustain year- cies infesting humans [27], and also the most abundant round transmission of tick-borne pathogens, and possi- tick species detected on passerine birds during migra- bly contribute to reseeding foci of infections in temperate tion in southern Sweden [2]. Tree species of potentially and boreal areas. Indeed, several studies in the Scandi- zoonotic Babesia species—B. divergens, B. microti, and navian countries have shown the presence of ticks and B. venatorum—have been recorded at prevalence rates tick-borne infections in returning migratory birds, espe- of 0.2%, 3.2%, and 1.0%, respectively, in questing I. rici- cially well-studied pathogens such as Borrelia burgdor- nus ticks in southern Sweden [28]. In a study that exam- feri sensu lato (s.l.) [2], Rickettsia spp. [3], and tick-borne ined 2038 I. ricinus ticks which had been removed from encephalitis (TBE) virus [4]. However, the potential for humans in Sweden and on the Åland Islands, Finland, B. less-studied, rarer, or unknown pathogens in bird-borne capreoli, B. microti, and B. venatorum were recorded in ticks to be detected in birds has come into focus recently, 0.25%, 1.60%, and 1.30% of the ticks, respectively [29]. for instance with the bacterium Neoehrlichia mikurensis, Te aim of this study is to better understand the poten- which once identifed has been shown to be fairly abun- tial role of migratory birds as disseminators of Babesia dant in ticks [5]. Another pathogen of concern is Babesia protozoan parasites and to determine the prevalence of spp., a protozoan parasite causing babesiosis—an emerg- Babesia spp. in ticks infesting birds during their spring ing tick-borne human disease in the Holarctic region. and autumn migrations in south-eastern Sweden. Part of More than 100 species of Babesia have been described. this study, related to data on the prevalence of Borrelia Te majority have been recorded in mammals, and 16 spp. and the TBE virus in ticks removed from migratory species have so far been described from avian hosts [6, birds during 2009, has been reported elsewhere [30]. 7]. In Europe, the roe deer (Capreolus capreolus) is con- sidered the main vertebrate host of both Babesia capreoli Methods and Babesia venatorum [8, 9], while Babesia divergens Sampling, analyses, and processing of ticks is prevalent in cattle in southern Sweden [10, 11]. Sero- Full details of the sampling site, procedure for bird cap- logical studies indicate that another species of Babe- tures and bird classifcation, determination of devel- sia—Babesia motasi—may be common in sheep herds opmental stage and species of collected ticks, and total in south-eastern Sweden [12]. Several of the mammal- nucleic acid extraction from ticks and cDNA synthesis associated species, such as B. divergens, Babesia duncani, are available in the previous report [30]. B. venatorum, and Babesia microti, are zoonotic patho- In short, ticks were collected from birds captured gens and of increasing medical importance, causing from during the periods 15 March–15 June and 15 July–15 asymptomatic infections to mild or serious, sometimes November 2009 at the Ottenby Bird Observatory, which even fatal, human disease, particularly in immunocom- is located on the southern point of the island of Öland promised persons [13–18]. In Europe, most of the severe in south-eastern Sweden (56° 12′ N, 16° 24′ E). Trapped cases of human babesiosis have involved infections of B. birds were identifed to species level and classifed into divergens [14, 15, 18–20]. However, the relatively recent residents, short-distance migrants, partial migrants, and discovery of the closely related B. venatorum [21, 22] may long-distance migrants. Any collected tick was photo- suggest that some of the earlier cases diagnosed as due to graphed and morphologically identifed to stage of devel- B. divergens may in fact have been caused by B. venato- opment (larva, nymph, or adult), and sex of adults. Each rum. During the last decades, the recorded incidence of tick was individually homogenized using a TissueLyser II human babesiosis due to B. microti, which is present in a (Qiagen) followed by extraction, purifcation, and isola- reservoir of small mammals in the Holarctic region, has tion of total nucleic acids using ­MagAttract® Viral RNA increased considerably in the north-eastern United States M48 kit in a BioRobot M48 workstation (Qiagen). Te [23]. A few cases of human disease caused by B. microti total nucleic acids were reverse-transcribed to cDNA or B. microti-like species have recently also been diag- using the illustra™ Ready-to-Go RT-PCR Beads kit (GE nosed in Europe and Asia [19, 24]. Healthcare, Amersham Place, UK), which served as tem- In nature, most mammalian Babesia spp. are transmit- plate in all the polymerase chain reaction (PCR) assays. ted by ixodid ticks [7, 25]. However, there is circumstan- To identify the genus and species of the ticks, each tial evidence suggesting that some of the avian Babesia specimen was analysed by a PCR method targeting the spp. may be vectored by soft ticks [7]. In North Amer- tick mitochondrial 16S rRNA gene followed by DNA ica, blood transfusion is recognized as an increasing and sequencing, as previously described [30]. Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 3 of 10

Sampling of birds was approved by the Swedish Board positions at the 18S rRNA gene, specifcally on positions of Agriculture, delegated through the Animal Research 631, 663, and 1637 (99.83% nucleotide similarity) [32]. Ethics Committee in Linköping (decision 43–09). Te two frst positions are included in the DNA fragment amplifed by the primers used in this study. Additional Detection and determination of Babesia species fle 1 shows all the aligned Babesia nucleotide sequences. Detection of Babesia spp. was done using a SYBR green real-time PCR assay, as previously described [10]. Prim- Statistical analyses ers BJ1 (5′–GTC TTG TAA TTG GAA TGA TGG–3′) Data were presented as percentages for categorical vari- and BN2 (5′–TAG TTT ATG GTT AGG ACT ACG–3′) ables. Te categorical variables were analysed using the were designed to target the Babesia 18S rRNA gene to chi-square test, but when the expected frequency was < 5 amplify a 411–452-bp long amplicon depending on the in at least one of the cells of the contingency table, species of Babesia [31]. Fisher’s exact test was used. Statistical analyses were ™ A 20-μl reaction consisted of 10 µl SYBR Green PCR performed using GraphPad Prism version 8.0.0 for Win- Master Mix (Termo Fisher Scientifc, Stockholm, Swe- dows (GraphPad Software, San Diego, CA, USA). P val- den), 0.4 µl of each primer (10 µM; Invitrogen), 6.2 µl ues ≤ 0.05 were considered statistically signifcant. RNase-free water, and 3 µl cDNA template. Te PCR template of 3 µl consisted of a pool of cDNA from three Results tick specimens per reaction. A positive PCR control, con- Ticks collected from birds and ticks available for PCR sisting of 3 µl B. microti DNA (10 ng/µl) extracted from analyses an I. ricinus tick collected in Slovakia, was included in A total of 4601 bird individuals (4788 bird captures) of each run. Te B. microti DNA was kindly provided by 65 species were examined for ticks at least once during Dr Bronislava Víchová (Institute of Parasitology, Slo- the study period at the Ottenby Bird Observatory. A total vak Academy of Sciences, Slovakia) through Dr Martin of 749 bird individuals (759 bird captures) of 35 species Andersson (Centre for Ecology and Evolution in Micro- were infested with a total of 1339 ticks (Table 1). Tese bial Model Systems, Linnaeus University, Kalmar, Swe- results were previously reported in the study of Wil- den). As a negative control in the PCR assay, RNase-free helmsson et al. [30]. water was used as template. When Babesia-positive pools In this study, 1102 ticks (i.e., cDNA samples) were were detected, the samples were re-analysed individually. ™ available for analysis. Of these, 543 were larvae, 552 Te PCR reactions were performed on a C1000 Ter- ™ nymphs and two ticks were adult females. Five ticks mal Cycler, CFX96 Real-Time PCR Detection System could not be determined to developmental stage due (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using an to lack of photos. Te ticks available for analysis were activation step at 94 °C for 10 min, and 35 cycles of 94 °C molecularly identifed to: I. ricinus (n 1,051; 514 larvae, for 1 min, 55 °C for 1 min, and 72 °C for 2 min, and fnally = 532 nymphs, 5 specimens of unknown developmental one cycle of 72 °C for 5 min. Immediately after comple- stage), I. frontalis (n 24; 8 larvae, 15 nymphs, 1 adult tion of PCR, melting curve analyses were performed by = female), Haemaphysalis punctata (n 12; 12 larvae), and heating to 95 °C for 15 s, followed by cooling to 60 °C for = 1 Hyalomma marginatum (n 4; 1 larva, 2 nymphs, 1 adult 1 min, and subsequent heating to 95 °C at 0.8 °C ­min− = female). Te remaining 11 ticks could not be molecularly with continuous fuorescence recording. identifed to species due to unreadable sequences despite To determine the species of Babesia in the PCR-pos- several sequencing attempts. Instead, these 11 ticks itive samples, nucleotide sequencing of the PCR-prod- were only determined to genus level, Ixodes (8 larvae, 3 ucts was performed by Macrogen Inc. (Amsterdam, Te nymphs), based on the photos. Netherlands). All sequences obtained were confrmed by sequencing both strands. Te obtained chromatograms were initially edited and analysed using BioEdit Software Prevalence of Babesia species in ticks removed from birds v7.0 (Tom Hall, Ibis Terapeutics, Carlsbad, CA, USA), Of the ticks analysed, 2.4% (26/1,102) were positive for and the sequences were examined using the Basic Local Babesia spp. All Babesia-positive ticks belonged to I. rici- Alignment Search Tool (BLAST). Sequences obtained nus (26/1,051). Of these, 2.0% of the I. ricinus larvae were have been deposited in GenBank with accession numbers Babesia-positive in the PCR-assay (10/514) and 3.0% of ranging from MW554592 to MW554617. the I. ricinus nymphs were Babesia-positive (16/532). Species determination of B. microti and B. venatorum All fve I. ricinus ticks that could not be determined to is possible by sequencing the amplicon from the real- developmental stage were Babesia-negative. No signif- time PCR assay. B. capreoli is highly similar to B. diver- cant diference in Babesia prevalence between larvae and gens, and the two species difer only at three nucleotide nymphs was detected. Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 4 of 10 ND 4 2 2 1 75 1 6 3 1 3 5 1 2 13 Hy. m 1L 1A 2 N H. p 1L 4L 1L 1L 1L 4L c stage I. f 5L, 11 N, 1A 1L 1L I. r 1 N 4 N 1 N 52L, 33 N 7 N 2L 2 N 1 N 278L, 194 N, 3U 4L, 2 N 1L 1L, 1 N 3 N 20L, 9 N 1 N 2L 2 N 1 N 2 N 29L, 32 N 2 N 19L, 30 N 3L, 19 N 2L, 7 N 2 N 2L, 1 N 7L, 2 N 23L, 17 N 59L, 31 N, 1U Tick species and developmental ­ species and developmental Tick spp. Ixodes 4L 38L, 13 N 1 N 1L, 1 N 2 N 13L, 2 N d Median no. ticks per Median no. (IQR) bird infested – 1 (1.00–2.00) – 2 (1.00–4.25) – 3.5 2 1.5 1 (1.00–3.25) 1 (1.00–2.00) 1 (1.00–2.00) – 2 1 (1.00–1.00) 2 (1.75–3.25) 1 – – 1 (1.00–6.00) – 1 1 (1.00–2.00) 1 (1.00–1.00) 1 (1.00–2.00) 1 (1.00–1.00) 1 (1.00–1.00) 1 (1.00–1.00) 1 (1.00–1.00) 1 (1.00–1.00) 1 (1.00–2.00) 1 (1.00–2.00) d ± ­ SE ± 0.33 ± 0.73 ± 1.50 ± 0.00 ± 0.50 ± 0.75 ± 0.09 ± 0.21 ± 0.00 ± 0.00 ± 0.78 ± 0.00 ± 1.67 ± 0.00 ± 0.22 ± 0.00 ± 0.14 ± 0.14 ± 0.00 ± 0.00 ± 0.00 ± 0.10 ± 0.37 ± 0.20 – – – – – – – Mean no. ticks per Mean no. bird infested 1.3 3.6 3.5 2.0 1.5 1.6 1.7 1.3 1.0 1.0 2.9 1.0 2.7 1.0 1.6 1.0 1.5 1.5 1.0 1.0 1.0 1.1 1.8 1.7 eastern Sweden, 2009 [ 30 ] eastern Sweden, 1 4 1 1 7 4 3 7 8 1 2 3 2 3 1 8 1 2 3 9 3 4 93 29 64 57 24 11 49 619 120 No. of ticks No. No. of infested No. (%) birds 1 (20) 3 (14) 1 (9.0) 26 (67) 1 (6.7) 2 (5.6) 2 (7.7) 2 (7.1) 4 (6.9) 368 (24) 6 (16) 1 (13) 2 (4.0) 3 (7.0) 10 (56) 2 (7.1) 1 (4.3) 1 (17) 3 (6.8) 1 (6.3) 2 (20) 40 (30) 3 (3.8) 38 (4.7) 16 (36) 9 (2.8) 3 (21) 4 (6.9) 10 (3.7) 27 (22) 72 (28) 5 8 6 21 11 39 15 36 26 28 58 37 50 43 18 28 23 44 16 10 79 44 14 58 134 810 319 270 123 254 No. of bird No. captures 1551 a Migratory ­ category PM LM LM LM SM SM PM SM PM SM SM SM LM LM LM LM LM LM PM R LM LM LM LM SM PM SM LM LM LM PM Ticks collected from bird species at Ottenby Bird Observatory, species at Ottenby Bird bird collected south - from Ticks 1 Table species Bird Accipiter nisus Accipiter Acrocephalus palustris Acrocephalus Acrocephalus scirpaceus Acrocephalus Anthus trivialis Carduelis cannabina Carduelis Carduelis fammea Carduelis Certhia familiaris Emberiza schoeniclus Emberiza Cyanistes caeruleus Cyanistes Erithacus rubecula Fringilla coelebs Fringilla Fringilla montifringilla Fringilla Hippolais icterina Lanius collurio Lanius Luscinia luscinia Luscinia Luscinia svecica Luscinia Motacilla alba Oenanthe oenanthe Parus major Parus Passer domesticus Passer Phoenicurus ochruros Phoenicurus Phoenicurus phoenicurus Phoenicurus Phylloscopus collybita Phylloscopus Phylloscopus trochilus Phylloscopus Prunella modularis Prunella Regulus regulus Sturnus vulgaris Sylvia atricapilla Sylvia curruca Sylvia communis Troglodytes troglodytes Troglodytes Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 5 of 10 ND 3 23 Hy. m H. p c stage I. f 1 N 1L, 2 N 1 N I. r 1 N 1L, 18 N 8L, 108 N, 1U 2L, 17 N Tick species and developmental ­ species and developmental Tick spp. Ixodes 6 N 2 N d Median no. ticks per Median no. (IQR) bird infested – 2 (1.00–4.50) 1 (1.00–3.00) 2 (1.00–3.50) d ± ­ SE ± 0.69 ± 0.25 ± 0.58 – Mean no. ticks per Mean no. bird infested 2.6 2.3 2.4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 22 149 No. of ticks No. 1 (100) No. of infested No. (%) birds 0 9 (31) 0 66 (45) 0 9 (15) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 5 9 2 1 1 1 2 8 1 1 2 1 1 2 1 2 29 23 60 15 16 43 23 31 25 55 37 19 147 No. of bird No. captures a Migratory ­ category LM SM LM PM SM SM SM SM SM LM PM R LM PM SM LM LM LM LM LM LM LM LM R LM LM R PM LM SM (continued) b 1 Table species Bird Acrocephalus arundinaceus Acrocephalus Turdus iliacus Turdus Acrocephalus schoenobaenus Acrocephalus Turdus merula Turdus Anthus pratensis Turdus philomelos Turdus Carduelis carduelis Carduelis Unknown Carduelis chloris Carduelis Carduelis spinus Carduelis Carpodacus erythrinusCarpodacus Coccothraustes coccothraustes Coccothraustes Corvus monedula Delichon urbica Dendrocopos major Dendrocopos Emberiza citrinella Emberiza Ficedula albicollis Ficedula Ficedula hypoleuca Ficedula Ficedula parva Ficedula Hirundo rustica Jynx torquilla Locustella naevia Locustella Motacilla fava Muscicapa striata Muscicapa Passer montanus Passer Phylloscopus borealis Phylloscopus Phylloscopus sibilatrix Phylloscopus Picus viridis Picus Pyrrhula pyrrhula Saxicola rubetra Serinus serinus Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 6 of 10 ND 145 Hy. m 1L, 2 N, 1A H. p 12L c stage I. f 8L, 15 N, 1A I. r 515L, 551 N, 5U Tick species and developmental ­ species and developmental Tick spp. Ixodes 56L, 27 N d Median no. ticks per Median no. (IQR) bird infested d ± ­ SE – Mean no. ticks per Mean no. bird infested 0 0 0 0 No. of ticks No. 1339 No. of infested No. (%) birds 0 0 0 0 749 1 1 1 42 No. of bird No. captures 4788 a Migratory ­ category LM LM SM SM Lanius collurio Lanius (continued) IQR interquartile range error, SE standard partial migrants where individuals either migrate short within individuals either migrate PM partial distances where SM short-distance in Africa), migrants the majority in Europe, cases wintering where of individuals winter (in many LM long-distance migrants migrants not determined to genus, genus, to , ND not determined marginatum m., Hyalomma Hy. punctate, Haemaphysalis , H. p., , I. frontalis , I. f. ricinus Ixodes I. r. missing photos, stage due to U unknown developmental L larva A adult female, N nymph, Probably Probably

Europe or remain resident, R residents resident, or remain Europe 1 Table a Bird species Bird b c d species, or developmental stage due to missing photos of ticks and/or loss sample missing photos stage due to or developmental species, Sylvia borin Sylvia nisoria Turdus pilaris Turdus Turdus viscivorus Turdus Total number of specimens Total Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 7 of 10

Tree species of Babesia were identifed: B. venato- nymphs. In contrast to our results, Franke et al. [37] and rum (n = 15), B. microti (n = 10), and B. capreoli (n = 1). Hildebrandt et al. [34] recorded B. microti in I. ricinus lar- B. venatorum was detected in 9 larvae and 6 nymphs, B. vae removed from several passerine bird species in Ger- microti in 10 nymphs, and B. capreoli in 1 larva (Table 2). many. We are not aware of any other reports where tick Of all samples that were determined to Babesia spe- larvae removed from birds have been shown to harbour cies level (n = 26; Table 2), 17 were detected in ticks cap- B. microti. On the contrary, the diferent taxa included in tured in spring (15 March–15 June), and 9 were detected the B. microti complex are generally considered to have in ticks in late summer–autumn (15 July–15 November). diferent species of small mammals, specifcally rodents No signifcant diference was detected between the pro- and shrews, as their main vertebrate reservoirs [13, 19, portions of Babesia-positive ticks collected in spring 38–43]. It might be possible that the B. microti infec- (17/495) and Babesia-positive ticks collected in late sum- tions recorded by Franke et al. [37] and Hildebrandt et al. mer–autumn (9/556). [34] in larvae of I. ricinus were acquired by co-feeding transmission from nymphal or adult ticks that had pre- Bird species with ticks positive for Babesia species viously fed on B. microti-infected mammals. However, Babesia-positive I. ricinus ticks were removed from experimental evidence from North America supports eight bird species in spring or in late summer–autumn the notion that birds can act as reservoirs of B. microti: (Table 3): the common redstart (Phoenicurus phoenicu- Hersh and colleagues tested 10 North American mammal rus, n = 7), European robin (Erithacus rubecula, n = 7), species and four bird species for reservoir competency, common blackbird (Turdus merula, n = 5), Eurasian wren i.e., capacity to infect the main vector in North America, (Troglodytes troglodytes, n = 2), tree pipit (Anthus trivi- Ixodes scapularis, with B. microti. Tey found reservoir alis, n = 2), common starling (Sturnus vulgaris, n = 1), competence levels of > 17% in white-footed mouse (Pero- common whitethroat (Sylvia communis, n = 1), and lesser myscus leucopus), racoon (Procyon lotor), short-tailed whitethroat (Sylvia curruca, n = 1). shrew (Blarina brevicauda), and eastern chipmunk (Tamias striatus), and < 6% but > 0% in all other species, Discussion including all four bird species tested including three spe- So far, only a few studies have investigated tick-borne cies belonging to the Turdidae family [44]. Tere is sig- pathogens in ticks, which are blood-feeding on migratory nifcant genetic heterogeneity among strains of B. microti birds in northern Europe, and the current study adds new [41–43, 45]. Terefore, further genetic investigations are information to this subject. Our study is the frst one to necessary as well as studies to explore whether bird spe- report the presence of Babesia species in ticks collected cies even in Europe are systemically infected, competent from birds in Sweden. reservoirs (transmission hosts) for any of these taxa of “B. Te prevalence of Babesia spp. detected in this study microti”. (2.4%) is on a similar level as what has been reported in Te tick species H. punctata and Hy. marginatum ticks collected from birds in other countries in northern have been reported to harbour diferent Babesia species, Europe, i.e., Norway, northern Germany, and north-west- where B. motasi, B. bovis, B. bigemina, B. caballi and B. ern Russia (1.0–4.7%) [33–35]. In those studies, the Babe- major have been detected in H. punctata, and B. microti sia species were identifed as B. venatorum, B. microti, has been detected in Hy. marginatum [46–49]. A sero- and B. divergens. In our study, B. venatorum was the most logical survey of sheep indicated that B. motasi may be prevalent species and was detected in nymphs as well as present on the island of Gotland, south-eastern Sweden, in larvae of I. ricinus. Both B. venatorum [8] and B. diver- where it is presumably vectored by H. punctata [12]. To gens [36] are known to be transovarially transmitted. our knowledge, neither H. punctata nor Hy. marginatum Tus, their presence in tick larvae should not be taken as ticks removed from birds have been investigated previ- an indication that the pathogen was derived from feeding ously for the presence of Babesia species. However, in the upon the avian hosts. To elucidate that, one would need present study, only I. ricinus ticks were positive for Babe- to investigate the presence of Babesia spp. in blood taken sia. Tis is not surprising, given the overall low preva- directly from the birds. Our data suggest that B. capreoli lence of Babesia spp. detected in the I. ricinus ticks, and is also transovarially transmitted in I. ricinus ticks, since the small number of specimens of the other tick species it was present in a larval tick. Tis was expected, given analysed. the close phylogenetic relationship between B. divergens In this study, the Babesia-positive ticks were found on and B. capreoli and since it is a trait regarded as common eight common passerine bird species. Given the loca- to all members of the genus Babesia sensu stricto [19]. tion, all these individuals are considered to be on active B. microti, on the other hand, is not considered to migration. Four of the species—European robin (Eritha- be vertically transmitted [19], and was only found in cus rubecula), common starling (Sturnus vulgaris), Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 8 of 10

Table 2 Prevalence of Babesia species in Ixodes ricinus ticks removed from birds captured at the Ottenby Bird Observatory, Sweden 2009 Tick developmental stage No. of ticks examined­ a No. (%) of Babesia-positive No. of ticks containing Babesia species determined by nucleotide ticks sequencing Babesia venatorum Babesia microti Babesia capreoli

Larva 514 10 (2.0) 9 1 Nymph 532 16 (3.0) 6 10 Unknownb 5 0 (0.0) Total 1051 26 (2.5) 15 10 1 a All ticks were examined for Babesia spp. by a real-time PCR assay b Ticks could not be identifed to developmental stage due to missing photos

Table 3 Species of Babesia identifed in Babesia-positive, immature Ixodes ricinus ticks removed from bird species Bird species Babesia-positive ticks (spring/ Babesia venatorum (larvae/ Babesia microti (larvae/ Babesia capreoli autumn)a nymphs) nymphs) (larvae/nymphs)

Phoenicurus phoenicurus 7 (5/2) 2 (2/–) 4 (–/4) 1 (1/–) Erithacus rubecula 7 (5/2) 6 (4/2) 1 (–/1) Turdus merula 5 (2/3) 3 (1/2) 2 (–/2) Troglodytes troglodytes 2 (1/1) 2 (2/–) Anthus trivialis 2 (2/–) 2 (–/2) Sturnus vulgaris 1 (1/–) 1 (–/1) Sylvia communis 1 (1/–) 1 (–/1) Sylvia curruca 1 (–/1) 1 (–/1) Total 26 (17/9) 15 (9/6) 10 (–/10) 1 (1/–) a Babesia-positive I. ricinus ticks were removed from birds during 15 March–15 June and 15 July–15 November 2009 at Ottenby Bird Observatory, south-eastern Sweden

common blackbird (Turdus merula), and Eurasian wren animals suggests that the risk of encountering poten- (Troglodytes troglodytes)—are common and widespread tially Babesia-infective ticks exists. However, the risk to short-distance migrant (or partial migrant) species that humans resulting from these introductions remains spec- occur in a range of habitats, including in the vicinity of ulative, especially in relation to the enzootic occurrence humans, in gardens, forests, and pastures. Te other four of these pathogens. species are obligatory long-distance migrants wintering Two of the three Babesia species, i.e., B. venatorum in sub-Saharan Africa, predominantly West Africa, and and B. microti, detected in this study are previously include tree pipit (Anthus trivialis), common redstart known to cause human disease. In Europe, the most (Phoenicurus phoenicurus), common whitethroat (Sylvia common cause of clinical human babesiosis is B. diver- communis), and lesser whitethroat (Sylvia curruca), and gens, which typically is diagnosed in immunocompro- although common, they are less associated with human mised individuals and often gives rise to a severe illness settlements [50]. Interestingly, 17 of the bird individuals [16, 51]. Human disease caused by B. divergens has also infested by Babesia-positive ticks were captured during been reported in immunocompetent patients [52]. A few their northward spring migration, suggesting a potential cases of B. microti and B. venatorum infection have also role of these birds as disseminators of Babesia proto- been reported in Europe [21, 22, 24, 53]. In contrast to zoan parasites into Sweden. Although the prevalence of B. divergens, B. venatorum, and B. microti, B. capreoli is Babesia spp. was low, the sheer number of birds involved considered not to be human-pathogenic [32]. in migration would imply a signifcant number of intro- In southern Sweden, a prevalence of 2.5% for B. ductions of infected ticks on an annual basis. Quantify- microti and/or B. divergens antibodies among healthy ing this number would require larger sample sizes and a individuals was recorded, and an even higher seroprev- wider sampling of host species. However, the presence alence (16.3%) among seropositive Borrelia burgdorferi of infected ticks in common passerine species that occur sensu lato patients was detected [54]. Despite the high in habitats also frequented by humans and domesticated seroprevalence, only two cases of human babesiosis Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 9 of 10

have so far been reported in Sweden [18, 53]. Tus, the Declarations risk of developing severe babesiosis after an I. ricinus Ethics approval and consent to participate tick bite among healthy individuals in Sweden appears Sampling of birds was approved by the Swedish Board of Agriculture, to be low [29]. However, as pointed out previously by delegated through Animal Research Ethics Committee in Linköping (decision 43–09). Ethical approval is not required for this study, because the analyses others [19, 25, 54], we cannot exclude that this poten- were performed on cDNA from ticks, which were removed from birds in tially severe infection is underdiagnosed. accordance with the animal welfare guidelines and regulations.

Consent for publication All authors have given consent for publication of this manuscript in the Para- Conclusions sites & Vectors journal. Tis is the frst study showing the presence of B. vena- Competing interests torum, B. microti, and B. capreoli in ticks removed from PL has been an external scientifc expert to Valneva Austria GmbH, Vienna, birds in Sweden. Te study also reveals that zoonotic Austria. All other authors declare that they have no competing interests. Babesia species are present in ticks infesting migratory Author details birds in south-eastern Sweden, which could lead to dis- 1 Division of Infammation and Infection, Department of Biomedical and Clini‑ semination of these tick-borne microorganisms into cal Sciences, Linköping University, Linköping, Sweden. 2 Department of Clini‑ 3 new areas. cal Microbiology, Region Jönköping County, Jönköping, Sweden. Depart‑ ment of Parasitology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, Katowice, Poland. 4 Department of Organismal Supplementary Information Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden. The online version contains supplementary material available at https://​doi.​ 5 Center for Ecology and Evolution in Microbial Model Systems, Linnaeus org/​10.​1186/​s13071-​021-​04684-8. University, Kalmar, Sweden. 6 Zoonosis Science Center, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. 7 Division of Infectious Dis‑ eases, Department of Biomedical and Clinical Sciences, Linköping University, Additional fle 1: The aligned Babesia nucleotide sequences based on Linköping, Sweden. PCR-products. Received: 25 November 2020 Accepted: 15 March 2021

Acknowledgements We would like to thank the staf members at the Ottenby Bird Observatory for species identifcation of the birds and for collecting the ticks attached to the birds. We are also grateful to Matilda Karlsson for skilful laboratory assistance. References The frst author OP would like to thank Professor Krzysztof Solarz from the 1. Hasle G. Transport of ixodid ticks and tick-borne pathogens by migratory Department of Parasitology, Medical University of Silesia in Katowice, Poland, birds. Front Cell Infect Microbiol. 2013;3:48. for his support. This is contribution no. 319 from Ottenby Bird Observatory. 2. Olsen B, Jaenson TG, Bergstrom S. Prevalence of Borrelia burgdorferi sensu lato-infected ticks on migrating birds. Appl Environ Microbiol. Authors’ contributions 1995;61(8):3082–7. PL, PF, BO, and JW planned the study and organized the collection of ticks 3. Elfving K, Olsen B, Bergstrom S, Waldenstrom J, Lundkvist A, Sjostedt at the Ottenby Bird Observatory. TGTJ determined the developmental stage A, et al. Dissemination of spotted fever rickettsia agents in Europe by of each tick and sex of the adult ticks. OP and PW performed the laboratory migrating birds. PLoS ONE. 2010;5(1):e8572. analyses, processed the data, and wrote the manuscript together with TGTJ. All 4. Waldenstrom J, Lundkvist A, Falk KI, Garpmo U, Bergstrom S, Lindegren G, authors read and approved the fnal manuscript. et al. Migrating birds and tickborne encephalitis virus. Emerg Infect Dis. 2007;13(8):1215–8. Funding 5. Labbe Sandelin L, Tolf C, Larsson S, Wilhelmsson P, Salaneck E, Jaenson TG, Open access funding provided by Linköping University.. This research was sup‑ et al. Candidatus Neoehrlichia mikurensis in ticks from migrating birds in ported as part of NorthTick, an Interreg project supported by the North Sea Sweden. PLoS ONE. 2015;10(7):e0133250. Programme of the European Regional Development Fund of the European 6. Yabsley MJ, Vanstreels RET, Shock BC, Purdee M, Horne EC, Peirce MA, Union. This study was also supported by the EU Interreg ÖKS V program et al. Molecular characterization of Babesia peircei and Babesia ugwidiensis ScandTick Innovation, project ID 20200422, reference no. 2015–000167, and provides insight into the evolution and host specifcity of avian piroplas‑ by The Swedish Research Council Branch of Medicine (grant no. K2008- mids. Int J Parasitol Parasites Wildl. 2017;6(3):257–64. 58X-14631–06-3), Carl Tryggers stiftelse, Helge Ax:son Johnsons stiftelse, 7. Peirce MA. A taxonomic review of avian piroplasms of the genus Babesia Längmanska Kulturfonden, Magnus Bergvalls stiftelse, the Medical Research Starcovici, 1893 (Apicomplexa: Piroplasmorida: Babesiidae). J Nat Hist. Council of Southeast Sweden (FORSS-657881, FORSS-931010), and by the 2000;34(3):317–32. Division of Laboratory Medicine, Region Jönköping County. This work was 8. Bonnet S, Jouglin M, L’Hostis M, Chauvin A. Babesia sp EU1 from roe carried out under the auspices of ESGBOR (the European Study Group on deer and transmission within Ixodes ricinus. Emerg Infect Dis. 2007;13 Lyme Borrelioses) and VectorNet, a European network for sharing data on the (8):1208–10. geographic distribution of arthropod vectors, transmitting human and animal 9. Andersson MO, Bergvall UA, Chirico J, Christensson M, Lindgren PE, disease agents (framework contract OC/EFSA/AHAW/2013/02-FWC1) funded Nordstrom J, et al. Molecular detection of Babesia capreoli and Babesia by the European Food Safety Authority (EFSA) and the European Centre for venatorum in wild Swedish roe deer Capreolus capreolus. Parasit Vectors. Disease prevention and Control (ECDC). 2016;9:221. 10. Andersson MO, Víchová B, Tolf C, Krzyzanowska S, Waldenstrom J, Availability of data and materials Karlsson ME. Co-infection with Babesia divergens and Anaplasma The data supporting the conclusions of this article are included within the phagocytophilum in cattle (Bos taurus). Sweden Ticks Tick Borne Dis. article. Raw data can be shared with researchers upon a specifc request. 2017;8(6):933–5. Wilhelmsson et al. Parasites Vectors (2021) 14:183 Page 10 of 10

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